Target

Function

Necessary for the fragmentation of Golgi stacks during mitosis and for their reassembly after mitosis. Involved in the formation of the transitional endoplasmic reticulum (tER). The transfer of membranes from the endoplasmic reticulum to the Golgi apparatus occurs via 50-70 nm transition vesicles which derive from part-rough, part-smooth transitional elements of the endoplasmic reticulum (tER). Vesicle budding from the tER is an ATP-dependent process. The ternary complex containing UFD1L, VCP and NPLOC4 binds ubiquitinated proteins and is necessary for the export of misfolded proteins from the ER to the cytoplasm, where they are degraded by the proteasome. The NPLOC4-UFD1L-VCP complex regulates spindle disassembly at the end of mitosis and is necessary for the formation of a closed nuclear envelope (By similarity). Regulates E3 ubiquitin-protein ligase activity of RNF19A.

Immunofluorescent analysis of VCP using VCP Monoclonal antibody (5) ab11433 shows staining in HeLa cells. VCP staining (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing VCP ab11433 at a dilution of 1:20-1:200 over night at 4 ?C washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized Human colon carcinoma tissues. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:1000 with a mouse monoclonal antibody recognizing Anti-VCP ab11433 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

ab24762 detecting VCP in Human platelets by Flow Cytometry. Platelets were isolated by spinning Platelet rich plasma on Histopaque. Cells were fixed in PFA and permeabilized in 0.1% Triton-X100 in 2% BSA for 30 minutes. The primary antibody was diluted 1/400 in 2% BSA in PBS and incubated with the sample for 18 hours at 4°C. An Alexa Fluor® 488-conjugated Goat anti-Mouse IgG (H+L) antibody, diluted 1/500 was used as the secondary antibody.

Immunofluorescent analysis of VCP using VCP Monoclonal antibody (5) ab11433 shows staining in C6 glioma cells. VCP staining (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing VCP ab11433 at a dilution of 1:20-1:200 over night at 4 ?C washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized Human brain tissue tissues. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:1000 with a mouse monoclonal antibody recognizing Anti-VCP ab11433 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Immunofluorescent analysis of VCP using VCP Monoclonal antibody (5) ab11433 shows staining in WiDr colon carcinoma cells. VCP staining (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing VCP ab11433 at a dilution of 1:20-1:200 over night at 4 ?C washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

ab11433 (1µg/ml) staining VCP in human left ventricle using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear, cytoplasmic and cell membrane staining of cardiomyocutes and smooth muscle cells of cardiac artery.Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

ab11433 staining VCP in Human H1299 cells by Immunocytochemistry/ Immunofluorescence. The cells were paraformaldehyde fixed, permeabilized in 0.1% Triton X-100 and were blocked in 10% serum at 25°C for 1 hour. Primary antibody was used at 1/1000 dilution (PBS) and incubated with sample for 1 hour at 37°C. An Alexa Fluor® 488 conjugated Goat polyclonal to mouse IgG was used as secondary at 1/1000 dilution. Image shows green color staining with ab11433 and nuclei were stained blue with DAPI.